The use of Green Fluorescent Protein (GFP) derived from Aequorea Victoria has revolutionised research into many cellular and molecular-biological processes. GFP allows researchers to label proteins within cells with an intrinsic fluor, so obviating the requirement to perform chemical labelling of proteins, and allowing development of assays of biological processes in intact living cells.
U.S. Pat. No. 5,491,084 describes the use of GFP as a biological reporter. Early applications of GFP as a biological reporter (Chalfie et al. Science, (1994), 263, 802–5; Chalfie, et al, Photochem.Photobiol., (1995), 62(4), 651–6) used wild type (native) GFP (wtGFP), but these studies quickly demonstrated two areas of deficiency of wtGFP as a reporter for use in mammalian cells. Firstly, the protein being derived from a poikilothermic marine organism does not undergo protein folding efficiently when expressed in mammalian cells cultured at 37° C., resulting in weak fluorescence. Secondly, the spectral characteristics of the wtGFP are not ideally suited to use as a cellular reporter, requiring excitation with electromagnetic radiation in the near-UV range, which is potentially damaging to living cells.
Consequently, significant effort has been expended to produce variant mutated forms of GFP with properties more suitable for use as an intracellular reporter.
A number of mutated forms of GFP with altered spectral properties have been described. A variant-GFP (Heim et al. (1994) Proc.Natl.Acad.Sci. 91, 12501) contains a Y66H mutation which blue-shifts the excitation and emission spectrum of the protein. However, this protein is only weakly fluorescent and requires potentially damaging Lw excitation.
A further mutant of GFP (Heim et al, Nature, (1995), 373, 663–664) contains a S65T mutation which red-shifts the optimum excitation and emission wavelengths relative to wtGFP and which is 4–6 fold brighter than wtGFP when expressed as a recombinant protein at 25° C. However, this variant does not yield bright fluorescence when expressed in hosts cultured at 37° C.
Ehrig et al (FEBS Lett., (1995), 367,163–6) describe two mutations of GFP, T203I and E222G, which individually delete one of the excitation maxima of wtGFP. The E222G mutation deletes the near-UV excitation peak at 395 nm and produces a red-shift in the excitation peak at 475 nm to 481 nm. The emission peak for this mutant protein is at 506 nm.
WO96/27675 describes two variant GFPs, obtained by random mutagenesis and subsequent selection for brightness, which contain the mutations V163A and V163A+S175G, respectively. These variants were shown to produce more efficient expression in plant cells relative to wtGFP and to increase the thermotolerance of protein folding. The double mutant V163A+S175G was observed to be brighter than the variant containing the single V163A mutant alone; however this mutant exhibits an undesirable blue-shifted excitation peak.
A further mutant, termed cycle-3, generated by molecular evolution through DNA shuffling (Crameri, A. et al, Nature Biotechnology, (1996), 14, 315–9) is available commercially from Invitrogen Inc. Cycle-3-GFP contains three mutations (F99S+M153T+V163A) which increase whole cell fluorescence approximately 42 fold when compared with wtGFP. However, this mutant retains the near-UV excitation maximum of the wtGFP, making it less suitable as a reporter for use in living cells.
The above mutations effectively address some of the spectral deficiencies of wtGFP as a biological reporter in providing variant forms of GFP which are compatible with lower energy excitation and which emit at wavelengths compatible with detection instrumentation commonly in use for measuring biological reporters. However, such mutations do not address the problem of inefficient folding and chromophore formation when wtGFP or spectral variants are expressed in hosts requiring growth at temperatures significantly greater than ambient.
U.S. Pat. No. 6,172,188 describes variant GFPs wherein the amino acid in position 1 preceding the chromophore has been mutated to provide an increase of fluorescence intensity. Such mutations include F64I, F64V, F64A, F64G and F64L, with F64L being the preferred mutation. These mutants result in a substantial increase in the intensity of fluorescence of GFP without shifting the excitation and emission maxima. F64L-GFP has been shown to yield an approximate 6-fold increase in fluorescence at 37° C. due to shorter chromophore maturation time.
In addition to the single mutants or randomly derived combinations of mutations described above, a variety of mutant-GFPs have been created which contain two or more mutations deliberately selected from those described above and other mutations, and which seek to combine the advantageous properties of the individual mutations to produce a protein with expression and spectral properties which are suited to use as a sensitive biological reporter in mammalian cells.
One mutant, commonly termed EGFP, available commercially from Clontech Inc., contains the mutations F64L and S65T (Cormack, B. P. et al, Gene, (1996), 173, 33–38). These mutations when combined, confer an approximate 35-fold increase in brightness over wtGFP and the spectral characteristics permit excitation and detection of EGFP with commonly used fluorescein excitation (488 nm) and emission filters (505 nm–530 nm). EGFP has been optimised for expression in mammalian systems, having been constructed with preferred mammalian codons.
U.S. Pat. No. 6,194,548 discloses GFPs with improved fluorescence and folding characteristics at 37° C. that contain, at least, the changes F64L and V163A and S175G. A further mutant GFP containing the F64L, S65T and V163A mutations has been described (Cubitt, A. B. et al, Methods in Cell Biology, (1999), 58, 19–29).
U.S. Pat. No. 6,077,707 describes a blue fluorescent protein (BFP) containing the F64L mutation in combination with Y66H and U.S. Pat. No. 6,194,548 describes a further BFP containing the F64L, Y66H, Y145F and L236R substitutions.